Solid support having electrostatic layer and use thereof

ABSTRACT

It is intended to provide a solid support capable of immobilizing nucleic acid molecules in a high proportion, and with a high bond strength to nucleic acid molecules. The solid support comprises a substrate and, provided thereon, an electrostatic layer for electrostatically attracting nucleic acid molecules and functional groups capable of covalently binding to nucleic acid molecules.

TECHNICAL FIELD

The present invention relates to a support for immobilizing DNA or thelike and an immobilized nucleic acid molecule.

BACKGROUND ART

Conventionally, in JP-A-7-75544 and JP-A-7-303469, polymerase chainreaction (PCR) is reported as a method of synthesizing a nucleotidesequence from an existing sequence.

The polymerase chain reaction (PCR) is a method in which a target DNA isflanked by a pair of primers, a DNA polymerase is allowed to act on itrepeatedly, whereby the region flanked by the primers can becontinuously amplified.

By PCR, only the target sequence can be substantially correctlyamplified to produce a large number of copies, further an efficientamplification is possible in a short time, therefore, PCR is widely usedfor a variety of studies, assays, tests and the like in biochemical,medical fields, etc., at present.

It has been conventionally assumed that the principle of PCR is tocontrol temperature, and the reaction is performed by repeating heatingand cooling procedures (thermal cycle). More specifically, for example,by denaturing a double-stranded DNA molecule, which is the target foramplification, into complementary single strands at a high temperature,annealing a primer, which has been selected to be complementary to apart of the DNA, to the strand by cooling, and extending the DNA to thedownstream of the primer with a DNA polymerase by heating again, and soon, one cycle consisting of the steps of denaturing, annealing andextending is repeated multiple times, whereby a large amount ofdouble-stranded DNA can be amplified.

Specifically, it is necessary to repeat the thermal cycle, whichconsists of 1) raising the temperature of a sample to 95° C. in order todisrupt the hydrogen bond of the double-stranded DNA, 2) subsequently,lowering the temperature of the sample to 45° C. in order to recombinethe DNA to a primer for replication, 3) further, raising the temperatureof the sample to 74° C. in order to replicate the DNA by extending theprimer with a heat-resistant polymerase, a number of times. In such aDNA amplification reaction, the foregoing thermal cycle was carried outby putting a sample into a container made of synthetic resin or thelike, and accommodating this container into an aluminum block.

However, the foregoing thermal cycle consumed a lot of time, and it tookseveral hours to obtain a required amount of DNA. In addition, whenreaction is carried out by controlling the temperature (heating andcooling), there is a limit on changing the temperature in an instant,and changeover of each step cannot be performed smoothly, whereby theaccuracy of the nucleotide sequence to be amplified may be affected, orDNA other than the target may be replicated in some cases. Further, inorder to change the temperature rapidly, a special apparatus ortechnique is needed, therefore there are an economic problem such asinvestment in equipment and a technical problem.

In view of the foregoing problems, as a support capable of easilyimmobilizing DNA and suitable for replicating DNA by DNA amplificationreaction, in WO 00/22108, WO 02/12891 or JP-A-2002-82116, a solidsupport which comprises, on the surface of a substrate, asurface-treated layer and chemically modified layer having a functionalgroup capable of covalently binding to a nucleic acid moleculesequentially has been developed.

However, the amount of DNA immobilized on the foregoing solid supportand the bond strength to DNA are not always sufficient, therefore, theemergence of a solid support capable of immobilizing DNA in a higherproportion and with a higher bond strength to DNA has been awaited.

The object of the present invention is to provide a solid supportcapable of immobilizing nucleic acid molecules in a higher proportion,and with a higher bond strength to nucleic acid molecules.

DISCLOSURE OF THE INVENTION

The present inventors carried out intensive studies in order to solvethe foregoing problems, and as a result, found out that the immobilizedamount of nucleic acid molecules and the bond strength to nucleic acidmolecules are significantly improved by further providing anelectrostatic layer for electrostatically attracting nucleic acidmolecules on a solid support having a functional group capable ofcovalently binding to a nucleic acid molecule on a substrate, therebyaccomplishing the present invention.

More specifically, the present invention includes the followinginventions.

-   (1) A solid support having an electrostatic layer for    electrostatically attracting a nucleic acid molecule and a    functional group capable of covalently binding to a nucleic acid    molecule on a substrate.-   (2) The solid support according to the foregoing (1), in which the    surface of the substrate is surface-treated with at least one kind    selected from diamond, a soft diamond, a carbonaceous matter and    carbide.-   (3) The solid support according to the foregoing (1) or (2), in    which the electrostatic layer comprises an amino group-containing    compound which does not covalently bind to the substrate.-   (4) The solid support according to the foregoing (1) or (2), in    which the electrostatic layer is composed of an amino    group-containing compound which covalently binds to the substrate    and the amino group-containing compound has an amino group at the    terminus to which the substrate does not bind.-   (5) The solid support according to any one of the foreging (1) to    (3), which is obtained by depositing a compound having an    unsubstituted or monosubstituted amino group and a carbon compound    on the substrate and then introducing a functional group capable of    covalently binding to a nucleic acid molecule.-   (6) The solid support according to any one of the foregoing (1) to    (4), which is obtained by dipping the substrate in a solution    containing a compound having an unsubstituted or monosubstituted    amino group and then introducing a functional group capable of    covalently binding to a nucleic acid molecule.-   (7) The solid support according to the foregoing (6), in which the    compound having an unsubstituted or monosubstituted amino group is    polyarylamine.-   (8) The solid support according to any one of the foregoing (1) to    (7), in which the nucleic acid molecule is DNA.-   (9) An immobilized nucleic acid molecule, which comprises a nucleic    acid molecule immobilized on a solid support according to any one of    the foregoing (1) to (8).-   (10) A method of producing a solid support characterized by    depositing a compound having an unsubstituted or monosubstituted    amino group and a carbon compound on the substrate and then    introducing a functional group capable of covalently binding to a    nucleic acid molecule.-   (11) A method of producing a solid support characterized by dipping    the substrate in a solution containing a compound having an    unsubstituted or monosubstituted amino group and then introducing a    functional group capable of covalently binding to a nucleic acid    molecule.-   (12) A method of immobilizing a primer on a solid support according    to any one of (1) to (8), and hybridizing a nucleic acid molecule to    the primer, thereby extending a nucleic acid molecule complementary    to the nucleic acid molecule.-   (13) A method of detecting a nucleic acid molecule, which comprises    immobilizing a primer on a solid support according to any one of (1)    to (8), hybridizing a nucleic acid molecule to the primer, extending    a nucleic acid molecule complementary to the nucleic acid molecule    in the presence of a labeled nucleic acid and reading a signal    derived from the labeled nucleic acid incorporated into the    complementary nucleic acid molecule.-   (14) A method of amplifying a nucleic acid molecule by immobilizing    a primer on a solid support according to any one of (1) to (8),    hybridizing a nucleic acid molecule to the primer and subjecting it    to PCR reaction.-   (15) A method of amplifying DNA by immobilizing a primer on a solid    support according to any one of (1) to (8), hybridizing DNA to the    primer and performing reaction with a strand-displacing DNA    polymerase.-   (16) The method according to (13), which further comprises the step    of amplifying the nucleic acid molecule after hybridizing a nucleic    acid molecule to the primer.

BEST MODE FOR CARRYING OUT THE INVENTION

Examples of a material of the substrate to be used in the presentinvention include, for example, silicone, glass, fiber, wood, paper,ceramics, and plastic (e.g., polyester resin, polyethylene resin,polypropylene resin, ABS resin (acrylonitrile butadiene styrene resin),nylon, acrylic resin, fluorocarbon resin, polycarbonate resin,polyurethane resin, methylpentene resin, phenolic resin, melamine resin,epoxy resin, polyvinyl chloride resin).

In the case where the foregoing members are used as the material of thesubstrate, a surface-treated layer may not be provided, however, it ismore preferred that surface treatment be carried out in order to firmlyimmobilize a compound for introducing a functional group capable ofcovalently binding to a nucleic acid molecule on the substrate.

For the surface treatment, it is preferred to use any of syntheticdiamond, high-pressure synthetic diamond, natural diamond, a softdiamond (e.g., a diamond-like carbon), amorphous carbon, a carbonaceousmatter (e.g., graphite, fullerene or carbon nanotube), a mixturethereof, or a laminated product thereof. In addition, carbide such ashafnium carbide, niobium carbide, silicon carbide, tantalum carbide,thorium carbide, titanium carbide, uranium carbide, tungsten carbide,zirconium carbide, molybdenum carbide, chromium carbide or vanadiumcarbide may be used. The term of soft diamond here is used as acollective term of a partial diamond structure, which is a mixture ofdiamond and carbon, such as, so-called a diamond-like carbon (DLC), andthe mixing ratio thereof is not particularly limited.

As one example of the surface-treated substrate, a substrate in which afilm has been formed with a soft diamond on a slide glass isexemplified. It is preferred that such a substrate be produced by theionization deposition method in a mixed gas containing 0 to 99% byvolume of hydrogen gas and the balance of methane gas (100 to 1% byvolume) with a diamond-like carbon.

It is preferred that the thickness of the surface-treated layer be 1 nmto 100 μm.

The formation of the surface-treated layer on the substrate can becarried out by a known method such as the microwave plasma CVD (chemicalvapor deposition) method, ECRCVD (electric cyclotron resonance chemicalvapor deposition) method, IPC (inductive coupled plasma) method, DCsputtering method, ECR (electric cyclotron resonance) sputtering method,ion plating method, arc ion plating method, EB (electron beam)deposition method, resistance heating vapor deposition method,ionization deposition method, arc deposition method, laser depositionmethod.

Examples of the substrate to be used in the present invention include,not only the structure in which the surface-treated layer has beenformed as described above, but also synthetic diamond, high-pressuresynthetic diamond, natural diamond, a soft diamond (e.g., a diamond-likecarbon), amorphous carbon; metals such as gold, silver, copper,aluminum, tungsten and molybdenum; plastic (such as polyester resin,polyethylene resin, polypropylene resin, ABS resin, nylon, acrylicresin, fluorocarbon resin, polycarbonate resin, polyurethane resin,methylpentene resin, phenolic resin, melamine resin, epoxy resin,polyvinyl chloride resin); the one formed by mixing and combining powderof the foregoing metal, powder of ceramic or the like with the foregoingresin as a binder; the one obtained by sintering at a high temperature amaterial such as powder of the foregoing metal or powder of ceramic,which has been powder-pressed with press-molding machine. In addition,the substrate may be a laminated product or a composite of the foregoingmaterials (for example, a composite of diamond and another substance,(e.g. a two-phase substance)).

The shape and the size of the substrate are not particularly limited.However, with regard to the shape, it may be in the form of plate,thread, sphere, polygon, powder and the like, and with regard to thesize, in the case of using the one in the form of plate, it is generallyabout 0.1 to 100 mm of width, 0.1 to 100 mm of length and 0.01 to 10 mmof thickness.

In addition, on the front face or back face of the substrate, amonolayer of Ti, Au, Pt, Nb, Cr, TiC, TiN or the like, or a compositelayer thereof may be formed as a reflective layer. The thickness of thereflective layer is preferably 10 nm or more, more preferably 100 nm ormore, because it is necessary to be uniform throughout the surface.

In the case of using glass as the substrate, it is also preferred thatthe surface be intentionally roughened within the range of 1 nm to 1000nm expressed in Ra (JIS B 0601). Such roughened surface is advantageousin that the surface area of the substrate is increased, whereby a largeamount of DNA probes or the like can be immobilized at a high density.

The solid support of the present invention is provided with anelectrostatic layer for electrostatically attracting a nucleic acidmolecule.

The electrostatic layer is not particularly limited as long as itattracts a nucleic acid molecule electrostatically and improves theimmobilized amount of nucleic acid molecules, however, it can be formedby, for example using a positively charged compound such as an aminogroup-containing compound.

Examples of the foregoing amino group-containing compound include acompound having an unsubstituted amino group (—NH₂) or an amino group,which has been monosubstituted with an alkyl group having 1 to 6 carbonatoms or the like (—NHR; R is a substituent), and for example,ethylenediamine, hexamethylenediamine, n-propylamine, monomethylamine,dimethylamine, monoethylamine, diethylamine, arylamine, aminoazobenzene,aminoalcohol, (e.g., ethanolamine), acrinol, aminobenzoic acid,aminoanthraquinone, amino acids (glycine, alanine, valine, leucine,serine, threonine, cysteine, methionine, phenylalanine, tryptophan,tyrosine, proline, cystine, glutamic acid, aspartic acid, glutamine,asparagine, lysine, arginine and histidine), aniline, a polymer thereof(e.g., polyarylamine and polylysine) and a copolymer thereof; apolyamine (polyvalent amine) such as 4,4′,4″-triaminotriphenylmethane,triamterene, spermidine, spermin and putrescine.

The electrostatic layer may be formed without covalently binding to thesubstrate or the surface-treated layer, or may be formed by covalentlybinding to the substrate or the surface-treated layer.

In the case of forming the electrostatic layer without covalentlybinding to the substrate or the surface-treated layer, a carbonaceousfilm containing an amino group is formed by, for example, introducingthe foregoing amino group-containing compound into a film-formingapparatus when the surface-treated layer is formed. As the compound tobe introduced into a film-forming apparatus, ammonia gas may be used. Inaddition, the surface-treated layer may be a multiple layer in which alayer containing an amino group is formed after forming an adherentlayer. In this case, film forming may be carried out also in anatmosphere containing ammonia gas.

Further, in the case of forming the electrostatic layer withoutcovalently binding to the substrate or the surface-treated layer, it ispreferred to introduce a functional group capable of covalently bindingto a nucleic acid molecule after depositing the forgoing compound havingan unsubstituted or monosubstituted amino group and a carbon compound onthe substrate in order to enhance the affinity, namely adhesivenessbetween the electrostatic layer and the substrate or the surface-treatedlayer. The carbon compound to be used here is not particularly limitedas long as it can be supplied as a gas, however, preferred are, forexample, methane, ethane and propane that are gas at a normaltemperature. As the method for deposition, the ionization depositionmethod is preferred. As the condition of the ionization depositionmethod, it is preferred that the working pressure be in the range of 0.1to 50 Pa and the accelerating voltage be in the range of 200 to 1000 V.

In the case of forming the electrostatic layer by covalently binding tothe substrate or the surface-treated layer, the electrostatic layer canbe formed by, for example, irradiating the substrate or the substrateprovided with the surface-treated layer with ultraviolet rays inchlorine gas to chlorinate the surface, and reacting, among theforegoing amino group-containing compounds, for example, a polyvalentamine such as polyarylamine, polylysine4,4′,4″-triaminotriphenyl-methane or triamterene to introduce an aminogroup into the terminus to which the substrate does not bind.

Further, in the case of carrying out the reaction of introducing afunctional group capable of covalently binding to a nucleic acidmolecule into the substrate provided with the electrostatic layer (e.g.,introduction of a carboxyl group by using a dicarboxylic acid orpolyvalent carboxylic acid) in a solution, it is preferred to introducea functional group capable of covalently binding to a nucleic acidmolecule after dipping the substrate in a solution containing theforegoing compound having an unsubstituted or monosubstituted aminogroup. Examples of the solvent for the foregoing solution include, forexample, water N-methylpyrrolidone and ethanol.

In the case of introducing a carboxyl group by using a dicarboxylic acidor polyvalent carboxylic acid into the substrate provided with theelectrostatic layer, it is preferred to activate it withN-hydroxysuccinimide and/or a carbodiimide in advance, or to carry outthe reaction in the presence of N-hydroxysuccinimide and/or acarbodiimide.

In the case of forming the electrostatic layer by dipping the substratein a solution containing the compound having an unsubstituted ormonosubstituted amino group, if polyarylamine is used as the aminogroup-containing compound, the adhesiveness to the substrate will beexcellent and the immobilized amount of the nucleic acid molecule willbe more improved.

It is preferred that the thickness of the electrostatic layer be 1 nm to500 μm.

As described above, the surface of the substrate provided with theelectrostatic layer is chemically modified in order to introduce afunctional group capable of covalently binding to a nucleic acidmolecule.

Examples of the foregoing functional group include, for example, acarboxyl group, active ester group, haloformyl group, hydroxyl group,sulfate group, cyano group, nitro group, thiol group and amino group.

Examples of the compound to be used for introducing a carboxyl group asthe functional group include, for example, a halocarboxylic acidrepresented by the formula: X-R¹—COOH (wherein X represents a halogenatom, and R¹ represents a divalent hydrocarbon group having 1 to 12carbon atoms) such as chloroacetic acid, fluoroacetic acid, bromoaceticacid, iodoacetic acid, 2-chloropropionic acid, 3-chloropropionic acid,3-chloroacrylic acid or 4-chlorobenzoic acid; a dicarboxylic acidrepresented by the formula: HOOC—R²—COOH (wherein R² represents a singlebond or a divalent hydrocarbon group having 1 to 12 carbon atoms) suchas oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acidor phthalic acid; a polyvalent carboxylic acid such as polyacrylic acid,polymethacrylic acid, trimellitic acid or butanetetracarboxylic acid; aketo acid or aldehyde acid represented by the formula: R³—CO—R⁴—COOH(wherein R³ represents a hydrogen atom or a divalent hydrocarbon grouphaving 1 to 12 carbon atoms and R⁴ represents a divalent hydrocarbongroup having 1 to 12 carbon atoms); a monohalide of dicarboxylic acidrepresented by the formula: X—OC—R⁵—COOH (wherein X represents a halogenatom and R⁵ represents a single bond or a divalent hydrocarbon grouphaving 1 to 12 carbon atoms) such as monochloride succinate ormonochloride malonate; an acid anhydride such as phthalic acidanhydride, succinic acid anhydride, oxalic acid anhydride, maleic acidanhydride or butanetetracarboxylic acid anhydride.

With regard to the carboxyl group introduced as described above, activeesterification thereof can be carried out with a dehydration condensingagent such as cyanamide or carbodiimide (e.g.1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide) and a compound such asN-hydroxysuccinimide.

Examples of the compound to be used for introducing a haloformyl groupas the functional group include, for example, a dihalide of dicarboxylicacid represented by the formula: X—OC—R⁶—CO—X (wherein X represents ahalogen atom, and R⁶ represents a single bond or a divalent hydrocarbongroup having 1 to 12 carbon atoms) such as chloride succinate orchloride malonate.

Examples of the compound to be used for introducing a hydroxyl group asthe functional group include, for example, a hydroxy acid or phenol acidrepresented by the formula: HO—R⁷—COOH (wherein R⁷ represents a divalenthydrocarbon group having 1 to 12 carbon atoms).

Examples of the compound to be used for introducing an amino group asthe functional group include, for example, an amino acid.

The foregoing compound forms an amide bond by condensing the carboxylgroup with the amino group in the electrostatic layer.

Among the foregoing compounds, the polyvalent carboxylic acid such aspolyacrylic acid, polymethacrylic acid, trimellitic acid orbutanetetracarboxylic acid can be also used for improvinghydrophilicity.

On the solid support of the present invention, either of the nucleicacid molecules, DNA and RNA, can be immobilized. The number of bases inDNA or RNA is generally 1 to 200, preferably 5 to 150. In addition, inthe case of DNA, either a single-stranded or double-stranded chain canbe immobilized.

The solid support of the present invention can be used for an extensionreaction of a nucleic acid molecule such as DNA. In this case, first aprimer is immobilized on the solid support, and then a single-strandedor double-stranded DNA is hybridized. Thereafter, DNA complementary tothe DNA hybridized to the primer is extended by a DNA extensionreaction.

As the primer, a single-stranded or double-stranded nucleic acidmolecule whose length and sequence are known is used. The length is notparticularly limited, however, it is preferably 5 to 200 bases, morepreferably 10 to 100 bases. The method of immobilizing the primer is notparticularly limited, however, for example, a primer solution isprepared by dissolving a nucleic acid molecule in a buffer and the solidsupport of the present invention is dipped in the solution, whereby theprimer can be immobilized on the surface of the solid support. Theprimer can be immobilized by performing dipping at generally 0 to 98°C., preferably 4 to 50° C. for generally 1 minute to 24 hours,preferably 10 minutes to 1 hour. In this case, by washing the solidsupport after dipping it for a predetermined period of time, the primerthat is not immobilized can be removed. In addition, by using anapparatus called spotter, a wide variety of primer solutions can beimmobilized on the surface of the solid support. In the case of usingthe spotter, for example, after primer solutions are spotted on thesolid support with a spotter, baking is performed in a heated oven for apredetermined period of time, thereafter the washinging is performed,whereby the primer that is not immobilized is removed. By using aspotter, a wide variety of primers can be immobilized at differentregions on the solid support, whereby a large number of assays can becarried out at one time, therefore it is advantageous in the field ofdetecting nucleic acids, which requires enormous assays.

In the conventional solid support, the primer is detached by a heattreatment in an extension reaction in some cases. On the other hand, inthe solid support of the present invention, the primer is not detachedeven if heat is applied, and an extension reaction can be carried out inthe state in which the primer has been immobilized on the solid support.

During this extension reaction, a labeled nucleic acid is allowed to beincorporated, and after the extension reaction, by reading a signalderived from the label, it can be detected whether or not a specific DNAhas been hybridized to the primer and the extension reaction hasprogressed. Therefore, it can be determined whether DNA, which can behybridized to the primer immobilized on the support, is contained in thetested sample. Therefore, it can be a useful detection means in researchand medical practices.

The label is not particularly limited as long as it can be incorporatedinto a nucleic acid molecule, however, examples include a fluorescentlabel (CyDye such as Cy3 and Cy5, FITC, RITC, Rhodamine, TexasRed, TET,TAMRA, FAM, HEX, ROX and the like), a radiolabel (α-³²P, γ-³²P, ³⁵S andthe like) etc. In the case of using the fluorescence-labeled nucleicacid, it can be detected by making a fluorescent photograph of the solidsupport after the extension reaction.

The solid support of the present invention can be used in DNAamplification reaction. In the case of amplifying it by PCR reaction,for example, first a forward primer is immobilized on the solid support,a single-stranded or double-stranded DNA is hybridized, and then acomplementary strand DNA is extended by an enzymatic reaction. Further,by carrying out the steps of (1) annealing, (2) hybridization and (3)extension reaction continuously, so-called PCR reaction progresses.

In the conventional solid support, there was a problem that the primeris detached by a heat treatment in PCR reaction or the control ofthermal cycle could not be carried out well. However, in the solidsupport of the present invention, the primer is not detached even ifheat is applied. Further, since the reaction is carried out not in acontainer, but in the state in which DNA has been immobilized on thesolid support, the temperature in PCR reaction can be accuratelycontrolled, and there is little possibility to affect the accuracy ofthe nucleotide sequence to be amplified, or to replicate DNA other thanthe target, therefore, DNA can be amplified efficiently.

In the case of using the solid support of the present invention for DNAamplification by the foregoing PCR, it is preferred that the thermalconductivity of the solid support be 0.1 W/cm·K or higher, morepreferably 0.5 W/cm·K or higher, most preferably 1 W/cm·K or higher. Itis based on the fact that if the thermal conductivity of the solidsupport is high, the following property for heating and cooling issuperior in the case of carrying out PCR reaction or the like.

Specifically, it is preferred that as the substrate for producing thesolid support, in terms of the thermal conductivity, diamond or the onecoated with diamond or the like as the surface-treated layer on avariety of substrates be used.

Further, by combining the foregoing detection using the labeled nucleicacid with amplification by PCR, even in the case where only a smallamount of DNA which can be hybridized to the primer on the solid supportis contained in a sample, DNA is replicated as described above. As aresult, a large amount of DNA is hybridized to the primer on the solidsupport, and the complementary strand is extended, whereby the detectionsensitivity can be enhanced.

Alternatively, as the enzyme to be used for the extension reaction, astrand-displacing DNA polymerase is selected and a reverse primer isadded, whereby DNA can be amplified on the solid support at a constanttemperature without undergoing the thermal cycle. The strand-displacingDNA polymerase is a DNA synthase that can synthesize the complementarystrand continuously by dissociating the double-stranded region if thedouble-stranded region is present in the extending direction during theprocess of synthesizing a DNA strand complementary to a template DNA.

The strand-displacing DNA polymerase is not particularly limited,however, examples include, for example, BCA BEST DNA polymerase (TakaraBio), Phi29 DNA polymerase (Amersham Bioscience) and the like.

In another embodiment of the present invention, by using cDNAsynthesized from mRNA as a target, RNA, though it is indirect, can alsobecome a target.

In this case, cDNA is obtained from mRNA by utilizing a reversetranscription reaction, however, cDNA can be immobilized on the solidsupport at the same time when it is obtained. First, a reversetranscription primer is bound to the chemically modified region of thesolid support. As the primer, an oligo dT primer, a primer complementaryto a specific nucleotide sequence or a random 6-mer primer is generallyused, however, in particular, it is desired to use an oligo dT primercomprising a sequence, in which there are about 10 to 20 Ts (thymines)in a row, corresponding to the poly(A)+ sequence at the 5′-terminus ofRNA.

In the case of using the oligo dT primer, the poly(A) region at the5′-terminus of RNA to be used as the template is annealed. A reversetranscriptase is allowed to act on this, and dNTP complementary to thetemplate RNA is polymerized at the 3′-terminus of the primer one afteranother, whereby cDNA is synthesized in the direction from the 5′ to the3′. The binding of primer, annealing, and polymerization of acomplementary strand by a reverse transcriptase in this reversetranscription reaction can be carried out by controlling the temperature(thermal cycle) according to the usual methods.

As described above, immobilization on the solid support can be carriedout at the same time when reverse transcription reaction is carried out,therefore, according to the method of the present invention, so-calledRT-PCR (reverse transcript-PCR) can be efficiently carried out.Therefore, the present invention is also useful for quantifying mRNA.

Further, by immobilizing the terminal base of an oligonucleotide on aterminal hydroxyl group or terminal carboxyl group through a hydrogenbond with the use of the support of the present invention, and furtherimmobilizing DNA having a base sequence complementary to thisoligonucleotide, the resultant product can be used as a DNA librarychip. In addition, by immobilizing a nucleotide, oligonucleotide, DNAfragment or the like instead of DNA, the resultant product can be usedas a library.

By performing the detection as described above with the use of the solidsupport of the present invention, diagnosis of a disease can be alsoperformed.

EXAMPLES

Hereunder, the present invention will be explained with reference to theExamples, however, the present invention is not intended to be limitedthereto.

Example 1 Introduction of Amino Group-containing Compound into Chamberwhen Applying Surface-treated Layer to Substrate (1)

DLC layer was formed at a thickness of 10 nm on a slide glass of 25 mm(width)×75 mm (length)×1 mm (thickness) by the ionization depositionmethod, at an accelerating voltage of 0.5 kV, using a mixed gas of 95%by volume of methane gas and 5% by volume of hydrogen gas as material.Then, using methane gas as a carrier gas, at the rate of 5 cm³/minute,it was introduced into a chamber through the ethylenediamine incubatedat 15° C. At a working pressure of 2 Pa and an accelerating voltage of0.5 kV, using methane and ethylenediamine as material, a layerconsisting of C, N and H was formed at a thickness of 10 nm.

Then, after butanetetracarboxylic acid anhydride, as a polyvalentcarboxylic acid, was condensed with the amino group in thesurface-treated layer consisting of C, N and H formed with methane andethylenediamine as material, it was activated by being dipped in anactivation solution, in which 0.1 M1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide and 20 mMN-hydroxysuccineimide had been dissolved in 300 mL of 0.1 M phosphatebuffer (pH 6), for 30 minutes.

Then, About 1 nL of 500 bp Cy3-labeled double-stranded DNA, which hadbeen amplified by PCR using lambda DNA prepared at a concentration of0.1 μg/μL as a template, was spotted on the substrate using a microarraymaker. Then, after it was heated in an oven at 80° C. for 3 hours andwashed with 2×SSC/0.2% SDS, the intensity of fluorescence of the spottedDNA was measured.

As a result, the intensity of fluorescence was 36,050. Further, when theintensity of fluorescence was measured after performing washing with2×SSC/0.2% SDS at 95° C., it was 35,540, which was hardly decreased atall.

As a comparison, after a slide glass was dipped in an ethanol solutioncontaining 2% by weight of 3-aminoprolyltriethoxysilane for 10 minutes,it was taken out, washed with ethanol, and dried at 110° C. for 10minutes. Then, after succinic acid anhydride was condensed with thissubstrate to which an amino group was introduced, it was activated bybeing dipped in an activation solution, in which 0.1 M1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide and 20 mMN-hydroxysuccineimide had been dissolved in 300 mL of 0.1 M phosphatebuffer (pH 6), for 30 minutes. After 500 bp Cy3-labeled double-strandedDNA, which had been amplified by PCR using lambda DNA as a template, wasimmobilized in the same manner on the thus obtained substrate, thesubstrate was washed with 2×SSC/0.2% SDS. As a result, the intensity offluorescence was 23,500. Further, when the intensity of fluorescence wasmeasured after performing washing with 2×SSC/0.2% SDS at 95° C., it was23,000, which was hardly decreased at all.

In other word, by using a covalent bond type substrate that hardly hasan electrostatic layer, although DNA can be immobilized more firmly by acovalent bond, the intensity of a fluorescence signal was not increased.

Further, after 500 bp Cy3-labeled double-stranded DNA, which had beenamplified by PCR using lambda DNA as a template, was immobilized in thesame manner on a substrate that does not have an electrostatic layer(after 5% polyacrylic acid aqueous solution was applied on a slide glassand dried, ultraviolet rays were irradiated for 60 minutes to make itinsoluble. Then, it was activated by being dipped in an activationsolution, in which 0.1 M 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimideand 20 mM N-hydroxysuccineimide had been dissolved in 300 mL of 0.1 Mphosphate buffer (pH 6), for 30 minutes), the substrate was washed with2×SSC/0.2% SDS. As a result, though the polyacrylic acid layer wasdetached, the intensity of fluorescence was 26,220 in the region wherethe layer remained. Further, the polyacrylic acid layer was completelydetached when being washed with 2×SSC/0.2% SDS at 95° C.

Example 2 Introduction of Amino Group-containing Compound into Chamberwhen Applying Surface-treated Layer to Substrate (2)

To a slide glass of 25 mm (width)×75 mm (length)×1 mm (thickness) by theionization deposition method, using methane gas as a carrier gas, at therate of 5 cm³/minute, it was introduced into a chamber through theethylenediamine incubated at 15° C. At a working pressure of 2 Pa and anaccelerating voltage of 0.5 kV, using methane and ethylenediamine asmaterial, a layer consisting of C, N and H was formed at a thickness of20 nm.

Then, after polyacrylic acid, as a polyvalent carboxylic acid, wascondensed with the amino group in the surface-treated layer consistingof methane and ethylenediamine in the presence of 0.1 M1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, it was activated bybeing dipped in an activation solution, in which 0.1 M1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide and 20 mMN-hydroxysuccineimide had been dissolved in 300 mL of 0.1 M phosphatebuffer (pH 6), for 30 minutes.

Then, about 1 nL of 500 bp Cy3-labeled double-stranded DNA, which hadbeen amplified by PCR using lambda DNA prepared at a concentration of0.1 μg/μL as a template, was spotted on the substrate using a microarraymaker. Then, after it was heated in an oven at 80° C. for 3 hours andwashed with 2×SSC/0.2% SDS, the intensity of fluorescence of the spottedDNA was measured.

As a result, the intensity of fluorescence was 34,050. Further, when theintensity of fluorescence was measured after performing washing with2×SSC/0.2% SDS at 95° C., it was 33,500, which was hardly decreased atall.

As a comparison, after 500 bp Cy3-labeled double-stranded DNA, which hadbeen amplified by PCR using lambda DNA as a template, was immobilized inthe same manner on a commercially available electrostatic type substrate(manufactured by Matsunami Glass Ind., LTD.; a substrate which is aslide glass applied with aminosilane (a silane coupling agent)), thesubstrate was washed with 2×SSC/0.2% SDS. As a result, the intensity offluorescence was 35,460. Further, when the intensity of fluorescence wasmeasured after performing washing with 2×SSC/0.2% SDS at 95° C., it wasdecreased to 26,210.

Further, after 500 bp Cy3-labeled double-stranded DNA, which had beenamplified by PCR using lambda DNA as a template, was immobilized in thesame manner on a substrate that does not have an electrostatic layer(after 5% polyacrylic acid aqueous solution was applied on a slide glassand dried, ultraviolet rays were irradiated for 60 minutes to make itinsoluble. Then, it was activated by being dipped in an activationsolution, in which 0.1 M 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimideand 20 mM N-hydroxysuccineimide had been dissolved in 300 mL of 0.1 Mphosphate buffer (pH 6), for 30 minutes), the substrate was washed with2×SSC/0.2% SDS. As a result, though the polyacrylic acid layer wasdetached, the intensity of fluorescence was 26,220 in the region wherethe layer remained. Further, the polyacrylic acid layer was completelydetached when being washed with 2×SSC/0.2% SDS at 95° C.

Example 3 Formation of Electrostatic Layer by Post-treatment

DLC layer was formed at a thickness of 10 nm on a slide glass of 25 mm(width)×75 mm (length)×1 mm (thickness) by the ionization depositionmethod, at an accelerating voltage of 0.5 kV, using a mixed gas of 95%by volume of methane gas and 5% by volume of hydrogen gas as material.

Then, it was chlorinated by being irradiated with ultraviolet rays for30 minutes in chlorine gas. Then, the substrate was dipped in apolyacrylic amine aqueous solution (0.1 g/L), whereby an electrostaticlayer was formed.

Then, after polyacrylic acid, as a polyvalent carboxylic acid, wascondensed with the amino group in the electrostatic layer in thepresence of 0.1 M 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, itwas activated by being dipped in an activation solution, in which 0.1 M1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide and 20 mMN-hydroxysuccineimide had been dissolved in 300 mL of 0.1 M phosphatebuffer (pH 6), for 30 minutes.

Then, about 1 nL of 500 bp Cy3-labeled double-stranded DNA, which hadbeen amplified by PCR using lambda DNA prepared at a concentration of0.1 μg/μL as a template, was spotted on the substrate using a microarraymaker. Then, after it was heated in an oven at 80° C. for 3 hours andwashed with 2×SSC/0.2% SDS, the intensity of fluorescence of the spottedDNA was measured.

As a result, the intensity of fluorescence was 35,000. Further, when theintensity of fluorescence was measured after performing washingwith2×SSC/0.2% SDS at 95° C., it was 34,500, which was hardly decreasedat all.

As a comparison, after 5% polyacrylic acid aqueous solution was appliedto a slide glass on which DLC was formed at a thickness of 10 nm anddried, ultraviolet rays were irradiated for 60 minutes to make itinsoluble. Then, it was activated by being dipped in an activationsolution, in which 0.1 M 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimideand 20 mM N-hydroxysuccineimide had been dissolved in 300 mL of 0.1 Mphosphate buffer (pH 6), for 30 minutes. After 500 bp Cy3-labeleddouble-stranded DNA, which had been amplified by PCR using lambda DNA asa template was immobilized in the same manner, and washed with2×SSC/0.2% SDS, the polyacrylic acid layer was completely detached.

Example 4 Immobilization of Primer by Dipping Method

DLC layer was formed at a thickness of 100 nm on an Si substrate cutinto 3 mm square by the ionization deposition method, at an acceleratingvoltage of 0.5 kV, using a mixed gas of 95% by volume of methane gas and5% by volume of hydrogen gas as material. Then, methane gas and hydrogengas were replaced with ammonia gas atmosphere and aminization wascarried out for 10 minutes by the plasma method.

Then, after a polyvalent carboxylic acid (polyacrylic acid) wascondensed with the amino group introduced in the surface-treated layer,it was activated by being dipped in an activation solution, in which 0.1M 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide and 20 mMN-hydroxysuccineimide had been dissolved in 0.1 M phosphate buffer (pH6), for 30 minutes.

Then, the immobilization reaction was carried out by dipping the solidsupport at room temperature for 1 hour in a solution of a forward primer(22 bases) of the 500 bp lambda DNA prepared at a concentration of 0.1g/μL using sterile water. After the reaction, the solid support waswashed with a washing solution (2×SSC/0.2% SDS) twice and rinsed withsterile water.

The solid support, on which the forward primer had been immobilized, wasdipped in a solution of the 500 bp lambda DNA that is complementary tothe immobilized primer (0.025 μg/μL, buffer: 5×SSC/0.5% SDS/20%formamide). After it was incubated at 98° C. for 5 minutes, it wasincubated at 42° C. for 12 hours. After the reaction, the solid supportwas washed with a washing solution (2×SSC/0.2% SDS) twice and rinsedwith sterile water.

The solid support after hybridization was dipped in a reaction solution(1×Exbuffer/0.025 mM Cy3-dCTP/1.25 mM dNTP/0.25 U ExTaq) and incubatedat 42° C. for 6 hours. After the reaction, the solid support was washedwith a washing solution (2×SSC/0.2% SDS) twice and rinsed with sterilewater.

When the solid support after the washing was observed with afluorescence image scanner, a fluorescence signal by the extensionreaction was detected.

Example 5 Immobilization of Primer by Spotting Method (1)

DLC layer was formed at a thickness of 100 nm on an Si substrate cutinto 3 mm square by the ionization deposition method, at an acceleratingvoltage of 0.5 kV, using a mixed gas of 95% by volume of methane gas and5% by volume of hydrogen gas as material. Then, methane gas and hydrogengas were replaced with ammonia gas atmosphere and aminization wascarried out for 10 minutes by the plasma method.

Then, after a polyvalent carboxylic acid (polyacrylic acid) wascondensed with the amino group introduced in the surface-treated layer,it was activated by being dipped in an activation solution, in which 0.1M 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide and 20 mMN-hydroxysuccineimide had been dissolved in 0.1 M phosphate buffer (pH6), for 30 minutes.

Then, solutions of forward primers (22 bases, 10 samples) of the 500 bplambda DNA prepared at a concentration of 0.1 μg/μL using 20% DMSOsolution were spotted on the solid support by using a spotter. Then,after being placed in an oven heated at 80° C. for 1 hour, it was washedwith a washing solution (2×SSC/0.2% SDS) twice and rinsed with sterilewater.

The solid support, on which the primers had been immobilized, was dippedin a solution of the single-stranded 500 bp lambda DNA that iscomplementary to the immobilized primers (0.025 μg/μL, buffer:5×SSC/0.5% SDS/20% formamide) After it was incubated at 98° C. for 5minutes, it was incubated at 42° C. for 12 hours. After the reaction,the solid support was washed with a washing solution (2×SSC/0.2% SDS)twice and rinsed with sterile water.

The solid support after hybridization was dipped in a reaction solution(1×Exbuffer/0.025 mM Cy3-dCTP/1.25 mM dNTP/0.25 U ExTaq) and incubatedat 42° C. for 6 hours. After the reaction, the solid support was washedwith a washing solution (2×SSC/0.2% SDS) twice and rinsed with sterilewater.

When the solid support after the washing was observed with afluorescence image scanner, a fluorescence signal by the extensionreaction was detected.

Example 6 Immobilization of Primer by Spotting Method (2)

DLC layer was formed at a thickness of 10 nm on a slide glass of 25 mm(width)×75 mm (length)×1 mm (thickness) by the ionization depositionmethod, at an accelerating voltage of 0.5 kV, using a mixed gas of 95%by volume of methane gas and 5% by volume of hydrogen gas as material.Then, methane gas and hydrogen gas were replaced with ammonia gasatmosphere and aminization was carried out for 10 minutes by the plasmamethod.

Then, after a polyvalent carboxylic acid (polyacrylic acid) wascondensed with the amino group introduced in the surface-treated layer,it was activated by being dipped in an activation solution, in which 0.1M 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide and 20 mMN-hydroxysuccineimide had been dissolved in 0.1 M phosphate buffer (pH6), for 30 minutes.

Then, solutions of forward primers (22 bases, 10 samples) of the 500 bplambda DNA prepared at a concentration of 0.1 μg/μL using 20% DMSOsolution were spotted on the solid support by using a spotter. Then,after being placed in an oven heated at 80° C. for 1 hour, it was washedwith a washing solution (2×SSC/0.2% SDS) twice and rinsed with sterilewater.

On the solid support, 15 μL of a solution of the 500 bp lambda DNA thatis complementary to the immobilized primers (0.025 μg/μL, buffer:5×SSC/0.5% SDS/20% formamide) was placed and further overlaid with acover glass. After hybridization was carried out by incubating at 42° C.for 5 hours, the cover glass was washed out with 0.1×SSC and the solidsupport was washed with a washing solution (2×SSC/0.2% SDS) twice andrinsed with sterile water.

After hybridization, on the solid support, 15 μL of a reaction solution(1×Exbuffer/0.025 mM Cy3-dCTP/1.25 mM dNTP/0.25 U ExTaq) was placed andfurther overlaid with a cover glass. After an extension reaction wascarried out by incubating at 42° C. for 5 hours, the cover glass waswashed out with 0.1×SSC and the solid support was washed with a washingsolution (2×SSC/0.2% SDS) twice and rinsed with sterile water.

When the solid support after the washing was observed with afluorescence image scanner, a fluorescence signal by the extensionreaction was detected.

Example 7 Annealing, Hybridization, Extension Reaction

DLC layer was formed at a thickness of 100 nm on an Si substrate cutinto 3 mm square by the ionization deposition method, at an acceleratingvoltage of 0.5 kV, using a mixed gas of 95% by volume of methane gas and5% by volume of hydrogen gas as material. Then, methane gas and hydrogengas were replaced with ammonia gas atmosphere and aminization wascarried out for 10 minutes by the plasma method.

Then, after a polyvalent carboxylic acid (polyacrylic acid) wascondensed with the amino group introduced in the surface-treated layer,it was activated by being dipped in an activation solution, in which 0.1M 1-[2-(dimethylamino)propyl]-3-ethylcarbodiimide and 20 mMN-hydroxysuccineimide had been dissolved in 0.1 M phosphate buffer (pH6), for 30 minutes.

Then, solutions of forward primers (22 bases, 10 samples) of the 500 bplambda DNA prepared at a concentration of 0.1 μg/μL using 20% DMSOsolution were spotted on the solid support by using a spotter. Then,after being placed in an oven heated at 80° C. for 1 hour, it was washedwith a washing solution (2×SSC/0.2% SDS) twice and rinsed with sterilewater.

The 500 bp lambda DNA, which was complementary to one of the immobilizedprimers, was added to a reaction solution (1×Exbuffer/0.025 mMCy3-dCTP/1.25 mM dNTP/0.25 U ExTaq) to give a final concentration of0.025 μg/μL.

The reaction solution was placed in a PCR tube and further a reverseprimer was added. The solid support, on which the primers had beenimmobilized, was dipped in the reaction solution and a cycle consistingof annealing (94° C., 1 minute), hybridization (60° C., 1 minute) andextension reaction (72° C., 1 minute) was repeated 30 times. After thereaction, the solid support was washed with a washing solution(2×SSC/0.2% SDS) twice and rinsed with sterile water.

When the solid support after the washing was observed with afluorescence image scanner, a more intense fluorescence signal wasdetected than in the case where the reaction was carried out at aconstant temperature.

Further, when the solid support, with which the extension reaction hadbeen carried out, was put in a PCR reaction solution and PCR reactionwas carried out to amplify the 500 bp lambda DNA, the amplification of500 bp region was confirmed by electrophoresis.

Example 8 Amplification Reaction Using Strand-displacing DNA Polymerase

DLC layer was formed at a thickness of 100 nm on an Si substrate cutinto 3 mm square by the ionization deposition method, at an acceleratingvoltage of 0.5 kV, using a mixed gas of 95% by volume of methane gas and5% by volume of hydrogen gas as material. Then, methane gas and hydrogengas were replaced with ammonia gas atmosphere and aminization wascarried out for 10 minutes by the plasma method.

Then, after a polyvalent carboxylic acid (polyacrylic acid) wascondensed with the amino group introduced in the surface-treated layer,it was activated by being dipped in an activation solution, in which 0.1M 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide and 20 mMN-hydroxysuccineimide had been dissolved in 0.1 M phosphate buffer (pH6), for 30 minutes.

Then, solutions of forward primers (22 bases, 10 samples) of the 500 bplambda DNA prepared at a concentration of 0.1 μg/μL using 20% DMSOsolution were spotted on the solid support by using a spotter. Then,after being placed in an oven heated at 80° C. for 1 hour, it was washedwith a washing solution (2×SSC/0.2% SDS) twice and rinsed with sterilewater.

The solid support, on which the primers had been immobilized, was dippedin a solution of the 500 bp lambda DNA that is complementary to theimmobilized primers (0.025 μg/μL, buffer: 5×SSC/0.5% SDS/20% formamide).After it was incubated at 98° C. for 5 minutes, it was incubated at 42°C. for 12 hours. After the reaction, the solid support was washed with awashing solution (2×SSC/0.2% SDS) twice and rinsed with sterile water.

The solid support after hybridization was dipped in a reaction solution(BcaBEST DNA polymerase/20 mM Tris/10 mM MgCl₂), to which a reverseprimer had been added, and was incubated at 60° C. for 6 hours. Afterthe reaction, the solid support was washed with a washing solution(2×SSC/0.2% SDS) twice and rinsed with sterile water. The enzyme used isa strand-displacing DNA polymerase (manufactured by TAKARA).

When the solid support after the washing was observed with afluorescence image scanner, a fluorescence signal by the extensionreaction was detected.

Further, when the solid support, with which the extension reaction hadbeen carried out, was put in a PCR reaction solution and PCR reactionwas carried out to amplify the 500 bp lambda DNA, the amplification of500 bp region was confirmed by electrophoresis.

Example 9 Annealing, Hybridization, Extension Reaction (2)

DLC layer was formed at a thickness of 100 nm on an Si substrate cutinto 3 mm square by the ionization deposition method, at an acceleratingvoltage of 0.5 kV, using a mixed gas of 95% by volume of methane gas and5% by volume of hydrogen gas as material. Then, methane gas and hydrogengas were replaced with ammonia gas atmosphere and aminization wascarried out for 10 minutes by the plasma method.

Then, after a polyvalent carboxylic acid (polyacrylic acid) wascondensed with the amino group introduced in the surface-treated layer,it was activated by being dipped in an activation solution, in which 0.1M 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide and 20 mMN-hydroxysuccineimide had been dissolved in 0.1 M phosphate buffer (pH6), for 30 minutes.

Then, a 0.1 μg/μL forward primer solution (5′-GATGAGTTGTGTCCGTACAACT-3′(see the sequence number 1 in the sequence table), 22bases, 20% DMSO), a0.1 μg/μL solution (5′-GGTTATCGAAATCAGCCACAGCGCC-3′ (see the sequencenumber 2 in the sequence table), 20% DMSO) and a 0.05 μg/μLforward+reverse primer solution (20% DMSO) were spotted on the solidsupport by using a spotter. Then, after being placed in an oven heatedat 80° C. for 1 hour, it was washed with a washing solution (2×SSC/0.2%SDS) twice and rinsed with sterile water.

Reaction solution 1 (0.025 μg/μL lambda DNA/1 pmol/μL reverseprimer/1×Exbuffer/0.025 mM Cy3-dCTP/1.25 mM dNTP/0.25U ExTaq) was placedin a PCR tube and the solid support, on which the primers had beenimmobilized, was dipped in the reaction solution and a cycle consistingof annealing (94° C., 30 seconds), hybridization and extension reaction(68° C., 30 seconds) was repeated 30 times. After the reaction, thesolid support was washed with a washing solution (2×SSC/0.2% SDS) twiceand rinsed with sterile water.

When the solid support after the washing was observed with afluorescence image scanner, a more intense fluorescence signal wasdetected than in the case where the reaction was carried out at aconstant temperature.

Further, when the solid support, with which the extension reaction hadbeen carried out, was put in reaction solution 2 (1 pmol/μL forwardprimer/1 pmol/μL reverse primer/1×Exbuffer/1.25 mM dNTP/0.25 U ExTaq)and a cycle consisting of annealing (94° C., 30 seconds), hybridizationand extension reaction (68° C., 30 seconds) was repeated 30 times, theamplification of DNA fragment in the 500 bp region was confirmed byelectrophoresis.

Example 10 Immobilization of Primer by Dipping Method (2)

After a glass substrate cut into 3 mm square was dipped in a polyacrylicamine aqueous solution adjusted to 0.1% and a polyvalent carboxylic acid(polyacrylic acid) was condensed with the amino group introduced in thesubstrate surface, it was activated by being dipped in an activationsolution, in which 0.1 M 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimideand 20 mM N-hydroxysuccineimide had been dissolved in 0.1 M phosphatebuffer (pH 6), for 30 minutes.

Then, the immobilization reaction was carried out by dipping the solidsupport at room temperature for 1 hour in a solution of a forward primer(22 bases) of the 500 bp lambda DNA prepared at a concentration of 0.1μg/μL using sterile water. After the reaction, the solid support waswashed with a washing solution (2×SSC/0.2% SDS) twice and rinsed withsterile water.

The solid support, on which the forward primer had been immobilized, wasdipped in a solution of the 500 bp lambda DNA that is complementary tothe immobilized primer (0.025 μg/μL, buffer: 5×SSC/0.5% SDS/20%formamide). After it was incubated at 98° C. for 5 minutes, it wasincubated at 42° C. for 12 hours. After the reaction, the solid supportwas washed with a washing solution (2×SSC/0.2% SDS) twice and rinsedwith sterile water.

The solid support after hybridization was dipped in a reaction solution(1×Exbuffer/0.025 mM Cy3-dCTP/1.25 mM dNTP/0.25 U ExTaq) and incubatedat 42° C. for 6 hours. After the reaction, the solid support was washedwith a washing solution (2×SSC/0.2% SDS) twice and rinsed with sterilewater.

When the solid support after the washing was observed with afluorescence image scanner, a fluorescence signal by the extensionreaction was detected.

Comparative Example

After a glass substrate cut into 3 mm square was dipped in a3-aminopropyltriethoxysilane solution adjusted at a concentration of 4%with 95% ethanol, the surface was aminized by baking it in an oven at100° C. for 20 minutes.

Then, by dipping the substrate at room temperature for 1 hour in asolution of a forward primer (22 bases) of the 500 bp lambda DNAprepared at a concentration of 0.1 μg/μL using sterile water, theimmobilization reaction by electrostatic binding was carried out. Afterthe reaction, the substrate was washed with a washing solution(2×SSC/0.2% SDS) twice and rinsed with sterile water. In addition, asubstrate that had been dipped in a solution of a fluorescence-labeledforward primer (22 bases) of the 500 bp lambda DNA was also prepared.

The substrate, on which the forward primer had been immobilized, wasdipped in a solution of the 500 bp lambda DNA that is complementary tothe immobilized primer (0.025 μg/μL, buffer: 5×SSC/0.5% SDS/20%formamide). After it was incubated at 98° C. for 5 minutes, it wasincubated at 42° C. for 12 hours. After the reaction, the substrate waswashed with a washing solution (2×SSC/0.2% SDS) twice and rinsed withsterile water.

The substrate after hybridization was dipped in a reaction solution(1×Exbuffer/0.025 mM Cy3-dCTP/1.25 mM dNTP/0.25 U ExTaq) and incubatedat 42° C. for 6 hours. After the reaction, the substrate was washed witha washing solution (2×SSC/0.2% SDS) twice and rinsed with sterile water.

When the substrate after the washing was observed with a fluorescenceimage scanner, a fluorescence signal by the extension reaction was notdetected at all.

With regard to the substrate to which the fluorescence-labeled forwardprimer solution (22 bases) of the 500 bp lambda DNA had beenimmobilized, although a faint fluorescence signal was observed after theimmobilization reaction, a fluorescence signal was not detected at allin the observation of a fluorescence image after the same procedure wascarried out. This indicates that the primer immobilized on the substratewas detached during the procedure.

The intensities of fluorescence measured with a fluorescence imagescanner in the foregoing Examples 4 to 10 are summarized in thefollowing Table 1. TABLE 1 Intensities of Fluorescence Signal inExamples 4 to 10 Intensity of Fluorescence Signal Example Beforereaction Spot A Spot B 4 4500 9000 — 5 4500 9500 11000 6 2500 6000  75007 4500 21000 22500 8 4500 15500 16000 9 4500 20000 22000 10 2000 7000 —

INDUSTRIAL APPLICABILITY

On the solid support of the present invention, a larger amount ofnucleic acid molecules can be immobilized than on the conventional solidsupport, and it can be immobilized firmly through a covalent bond.Therefore, the detection sensitivity and reliability that were theproblems of the conventional DNA array can be improved. Further, sincean extension reaction can be performed in the state in which a nucleicacid molecule has been immobilized, or a nucleic acid molecule can beamplified by performing PCR reaction, it is possible to aim atgeneralizing a DNA array.

1. A solid support having an electrostatic layer for electrostaticallyattracting a nucleic acid molecule and a functional group capable ofcovalently binding to a nucleic acid molecule on a substrate.
 2. Thesolid support according to claim 1, wherein the surface of the substrateis surface-treated with at least one kind selected from diamond, a softdiamond, a carbonaceous matter and carbide.
 3. The solid supportaccording to claim 1 or 2, wherein the electrostatic layer comprises anamino group-containing compound which does not covalently bind to thesubstrate.
 4. The solid support according to claim 1 or 2, wherein theelectrostatic layer is composed of an amino group-containing compoundwhich covalently binds to the substrate and the amino group-containingcompound has an amino group at the terminus to which the substrate doesnot bind.
 5. The solid support according to claim 1, which is obtainedby depositing a compound having an unsubstituted or monosubstitutedamino group and a carbon compound on the substrate and then introducinga functional group capable of covalently binding to a nucleic acidmolecule.
 6. The solid support according to any one of claims 1 to 4,which is obtained by dipping the substrate in a solution containing acompound having an unsubstituted or monosubstituted amino group and thenintroducing a functional group capable of covalently binding to anucleic acid molecule.
 7. The solid support according to claim 6,wherein the compound having an unsubstituted or monosubstituted aminogroup is polyarylamine.
 8. The solid support according to claim 1,wherein the nucleic acid molecule is DNA.
 9. An immobilized nucleic acidmolecule, which comprises a nucleic acid molecule immobilized on a solidsupport according to claim
 1. 10. A method of producing a solid supportcharacterized by depositing a compound having an unsubstituted ormonosubstituted amino group and a carbon compound on the substrate andthen introducing a functional group capable of covalently binding to anucleic acid molecule.
 11. A method of producing a solid supportcharacterized by dipping the substrate in a solution containing acompound having an unsubstituted or monosubstituted amino group and thenintroducing a functional group capable of covalently binding to anucleic acid molecule.
 12. A method of immobilizing a primer on a solidsupport according to claim 1, hybridizing a nucleic acid molecule to theprimer, thereby extending a nucleic acid molecule complementary to thenucleic acid molecule.
 13. A method of detecting a nucleic acidmolecule, which comprises immobilizing a primer on a solid supportaccording to claim 1, hybridizing a nucleic acid molecule to the primer,extending a nucleic acid molecule complementary to the nucleic acidmolecule in the presence of a labeled nucleic acid and reading a signalderived from the labeled nucleic acid incorporated into thecomplementary nucleic acid molecule.
 14. A method of amplifying anucleic acid molecule by immobilizing a primer on a solid supportaccording to claim 1, hybridizing a nucleic acid molecule to the primerand subjecting it to PCR reaction.
 15. A method of amplifying DNA byimmobilizing a primer on a solid support according to claim 1,hybridizing DNA to the primer and performing reaction with astrand-displacing DNA polymerase.
 16. The method according to claim 13,which further comprises the step of amplifying the nucleic acid moleculeafter hybridizing a nucleic acid molecule to the primer.